1. Field of the Invention
The present invention relates generally to the fields of diagnostic imaging. More particularly, it concerns methods and apparatus for polarized reflectance spectroscopy that may be used to assess cellular and nuclear morphology in order to, for example, diagnose various conditions in various tissues.
2. Description of Related Art
Approximately 1,200,000 people will be diagnosed with cancer in 1999 resulting in approximately 563,000 deaths. The majority of these cancers will be of epithelial origin. Early detection of pre-invasive epithelial neoplasia has the potential to increase patient survival and improve quality of life. However, many of the currently available screening and detection techniques for epithelial pre-cancers do not provide adequate sensitivity and specificity; furthermore, many screening and detection methods require extensive training to yield adequate clinical results. Thus, more sensitive and cost-effective screening and diagnostic techniques are needed to identify curable pre-cancerous lesions.
Pre-cancers are characterized by increased nuclear size, increased nuclear/cytoplasmic ratio, hyperchromasia and pleomorphism, which currently can only be assessed through invasive, painful biopsy. Elastic light scattering spectroscopy may provide a non-invasive tool to assess nuclear morphometry. The wavelength dependence of elastic light scattering is determined by scatterer sizes and refractive indices. Previous work using suspensions of polystyrene spheres and Intralipid with optical properties similar to tissue has shown that the sizes of scattering particles can be estimated from elastic scattering spectra using Mie theory. Recently this principle was applied to estimate distribution of nuclear size in the epithelium of the esophagus. In these experiments, tissue was illuminated with unpolarized light, and the spectrum of reflected light was measured. The reflected light consisted of both singly scattered light originating from the epithelial cells as well as a much stronger multiply scattered component produced in the stroma, which was modulated by hemoglobin absorption. The contributions of the background were modeled and subtracted from the experimental reflectance spectra in order to extract the relatively weak single scattering produced by epithelial cells. Although this approach has demonstrated some utility, its accuracy unfortunately depends strongly on the ability of the model to describe the scattering and absorption properties of the stromal layer.
In view of the above, it would be highly desirable to develop experimental techniques that would allow the elastic light scattering of epithelial cells to be measured directly with less dependence upon modeling capabilities.
In one respect, the invention is a method for assessing the size of a scattering element of a sample. As used herein, xe2x80x9csizexe2x80x9d is meant to be read broadly to include, but not to be limited to, size distributions, mean sizes, mean diameters, etc. As used herein, xe2x80x9cscattering elementxe2x80x9d is meant to be read broadly to include any material that causes scattering of radiation. Polarized reflectance spectra of the sample are obtained. A depolarization ratio is calculated using the spectra, and the size of the scattering element is calculated using the depolarization ratio.
In other respects, calculating the size of the scattering element includes varying one or more Mie theory parameters to determine a best fit between the depolarization ratio and a combination of forward and backward scattering terms. The sample may be in vivo. The sample may be in vitro. The sample may include a cell, and the scattering element may include a cytoplasm. The sample may include a cell, and the scattering element may include a cell nucleus. The cell may include a cervical cell. The cell may include an oral mucosa cell.
In another respect, the invention is a method for assessing the refractive index of a scattering element of a sample. Polarized reflectance spectra of the sample are obtained. A depolarization ratio is calculated using the spectra, and the refractive index of the scattering element is calculated using the depolarization ratio.
In another respect, the invention is a method for assessing the size of cell nuclei of a sample. Polarized reflectance spectra of the sample are obtained. A depolarization ratio is calculated using the spectra. One or more Mie theory parameters are varied to determine a best fit between the depolarization ratio and a combination of forward and backward scattering terms, and the size of the cell nuclei is determined using the Mie theory parameters.
In another respect, the invention is a method for assessing the size of a scattering element of a sample. Primary radiation is generated from a source. The primary radiation is polarized to produce polarized primary radiation. The polarized primary radiation is directed to the sample to generate reflected radiation. The reflected radiation is directed through a polarizer to produce filtered reflected radiation. The polarizer is configured to select reflected radiation parallel and perpendicular to the polarization of the polarized primary radiation. The filtered radiation is detected. A depolarization ratio is calculated using the detected filtered radiation, and the size of the scattering element is calculated using the depolarization ratio.
In another respect, the invention is a computer readable media containing program instructions for assessing the size of a scattering element of a sample. The computer readable media includes instructions for calculating a depolarization ratio from polarized reflectance spectra of the sample and instructions for calculating the size of the scattering element using the depolarization ratio.
In another respect, the invention is an apparatus for assessing the size of a scattering element of a sample. The apparatus includes a primary radiation source, a first polarizer, a second polarizer, and a detector. As used herein, xe2x80x9cdetectorxe2x80x9d is meant to be read broadly to include associated detector elements such as, but not limited to, filters and the like. The first polarizer is configured to polarize the primary radiation according to a first orientation. The second polarizer is configured to polarize reflected radiation according to a second and third orientation to produce filtered reflected radiation. The second orientation is substantially parallel to the first orientation, and the third orientation is substantially perpendicular to the first orientation. The detector is configured to detect the filtered radiation to produce a polarized reflectance spectra of the sample.
In other respects, the primary radiation source may include a halogen lamp source. The primary radiation source may include a xenon flashlight. The detector may include a plurality of bandpass filters. The detector may include a liquid crystal tunable filter and a variable wave retarder. The detector may include a single grating spectrograph coupled to an intensified photodiode array detector.
In another respect, the invention is a probe for assessing the size of a scattering element of a sample. It includes a tubing, a fiber disc, a plurality of fibers, and a polarizing film. The fiber disc is coupled to the tubing. The plurality of fibers are coupled to the fiber disc, and the fibers include one or more excitation fibers and one or more collection fibers. The polarizing film is divided in at least two parts, a first part in operative relation with the one or more excitation fibers, and a second part in operative relation with the one or more collection fibers.
In other respects, the probe may include one excitation fiber and two collection fibers. The excitation fiber and one of the collection fibers may be in operative relation with the first part of the polarizing film, and the other collection fiber may be in operative relation with the second part of the polarizing film. The first part of the polarizing film may be configured for a parallel polarization orientation and the other part of the polarizing film may be configured for a perpendicular polarization orientation. The probe may also include an optical window configured to protect the polarizing film. The one or more collection fibers may be located symmetrically relative to the one or more excitation fibers. The probe may also include a lens positioned in front of the plurality of fibers; the lens may be configured to create an overlap between an area illuminated by the one or more excitation fibers and areas from which scattering is gathered by the one or more collection fibers.
These features and associated advantages will become apparent with reference to the following detailed description of specific embodiments in connection with the accompanying drawings, wherein like reference numerals have been applied to like elements.